STEREOCHEMISTRY IN CATALYTIC OXIDATION BY HEME ENZYMES
Part I GENERAL INTRODUCTION Myoglobin (Mb) functions as an oxygen storage and carrier protein in muscle. This protein has been one of the most intensively investigated hemoproteins as evident from the accumulated biochemical and spectroscopic data. It has protoporphyrin IX as a prosthetic group, and...
| Main Authors: | , , |
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| Format: | Thesis |
| Language: | English |
| Published: |
2001
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| Subjects: | |
| Online Access: | https://ir.soken.ac.jp/?action=repository_uri&item_id=212 http://id.nii.ac.jp/1013/00000212/ |
| Summary: | Part I GENERAL INTRODUCTION Myoglobin (Mb) functions as an oxygen storage and carrier protein in muscle. This protein has been one of the most intensively investigated hemoproteins as evident from the accumulated biochemical and spectroscopic data. It has protoporphyrin IX as a prosthetic group, and is the first protein structure determined to high resolution by X-ray crystallographic analyses. Thus, myoglobin has ever been serving as a model system for the study of structure-function relationships in heme proteins.Part II Chapter 1. The Role of Val68 (E11) on Oxidation Activity and Enantioselectivity of Sperm Whale Myoglobin To probe the role of the distal valine 68 (E11) in sperm whale myoglobin (Mb) on the oxidation activity, site-directed mutagenesis was performed. A series of Mb mutants, H64D/V68X Mbs, have been prepared by replacing Val-68 with Gly, Ala, Ser, Leu, Ile, and Phe in H64D Mb. All of the mutant proteins are stable enough to be purified except for the H640/V68G mutant. The oxidation of the substrate thioanisole by H640/V68X Mb-I besides H64D/V68S was monitored by stopped-flow spectrometer and the sulfoxidation rate constants increase in the order of Phe ≦ Val < Leu < Ala < Ile. The results suggest that the volume of hydrophobic residue at the 68 position influences the sulfoxidation activity. A similar pattern is observed for the catalytic sulfoxidation of thioanisole by H64D/V68X Mbs and H2O2. The dominant product in the catalytic sulfoxidation is the R isomer for the H64D/V68A and H64D/V68S mutants, with more than 84% enantiomeric excess (% ee). However, increasing the polarity of the distal pocket by substituting Ala-68 with Ser in H64D Mb decelerates the catalytic sulfoxidation rate by 2-fold. On the other hand, the H64D/V681 mutant affords dominantly the S isomer with the highest turnover rate up to 413 turnover/min. The substitution of Vat-68 with Leu has little effect on enantioselectivity in the catalytic sulfoxidation but increases the reactivity with H202. Both the value of % ... |
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